Preventives or remedies for infection, anti-endotoxin agents, vaccine adjuvants and growth promoters

ABSTRACT

A preventive or remedy for infection, an anti-endotoxin agents, a vaccine adjuvants and a growth promoter each comprising a sugar cane-derived extract as an active ingredient which agent is safe to man and animals. Also presented are foods and feeds comprising these agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to preventives or remedies for infection,anti-endotoxin agents, vaccine adjuvants and growth promoters for man oranimals.

The present invention relates also to foods or feeds to prevent orremedy infection of man or animals.

The present invention relates also to foods or feeds to prevent orremedy human or animal diseases caused by endotoxin.

The present invention relates also to foods or feeds functioning asadjuvants for vaccines for man or animals.

The present invention relates also to foods or feeds promoting human oranimal growth.

2. Description of the Prior Art

Recently, various diseases or many infectious diseases of man andanimals are believed to be caused by weakened immunological functions oran insufficiency of immunological functions. In man, for example,immunological functions are weakened or made insufficient by bronchialasthma, allergic disease, joint rheumatism, autoimmune disease,nutrition lesion, surgical operations, aging, cancer, transplant oforgan, or conception, resulting in concurrence of infectious diseasessuch as infectious diseases of respiratory organs, septicemia, andinfectious diseases of urinary tracts. To treat these diseases andinfectious diseases, many kinds of antibiotics are administered.However, when the antibiotic is continuously administered, its efficacybecomes weaker due to development of resistant bacteria which leads to arecently closed-up problem of hospital infection. Therefore, it isdesired not to depend only on antibiotics but to develop drugs or foodwhich enforce immunological functions themselves to thereby prevent ortreat infection and to decrease dosage of antibiotics.

In the livestock and fishing industries, a large scale raising orovercrowded raising is performed to raise domestic animals, poultry orcultured fish efficiently. Such raising environment causes stresses inthe animals and immune insufficiency in the animals' infancy, whichleads often to various kinds of infectious diseases. As a countermeasurefor this, high dosages of the antibiotics for treating or preventingdiseases are administered to the animals. However, such a high dosagenecessitates it in return to administer more kinds or other kinds ofantibiotics in order to cope with problems of residual antibiotics,increase of resistant bacteria, and diseases caused by resistantbacteria.

Generally, known preventives or remedies comprise a single component ora plurality of components having similar structures as an activeingredient which is(are) prepared by extracting, condensing orsynthesizing the components. Consequently, it is apprehended that a longtime or high dosage of the preventive or remedy causes side effects.

Substances activating immunity to thereby prevent infection were found,such as some of Bacillus subtilis, Lactobacillus bifidus, andClostridium. The immunity activating effect or infection preventiveeffect of the followings were reported: Egg albumen (Japanese PatentApplication Laid-Open No. H3-251573), a mixture of two or more selectedfrom egg albumen, bacteria and garlic (National Publication ofTranslation of PCT Application No. H8-509211), one or more selected fromRosa roxburghii, mugwort and cabbage and a mixture thereof (JPALaid-Open No. H6-116158), and Glycyrrhiza component (JPA Laid-Open No.H9-143-85).

It is known that one can use bagasse as a culture medium for fungi suchas shiitake mushrooms, fomes japonicus, straw mushrooms, hackberris, andmushrooms. Also reported are an anti-virus agent of which activeingredient is an extract from basidiomycetes (JPA Laid-Open No.H2-286623, JPA Laid-Open No. H4-66536) and an anti-virus substanceobtained by fractionating and purifying an aqueous extract fromcultivated substances obtained by culturing shiitake fungus on a mediumcomprising bagasse and rice bran (JPA Laid-Open No. H5-4929).

An anti-virus agent is know which comprises active ingredientpolysaccharides and cytokinin generated by basidiomysetes cultured onbagasse as a medium (JPA Laid-Open No. S55-157517). However, the sameinventor later filed another patent application on an anti-animal virusagent essentially comprising both polysaccharides and water solublelignin as active ingredients which are prepared by subjecting bagasse toenzyme activity or boiling bagasse, followed by extraction (JPALaid-Open No. S57-106624). However, data on an agent comprising, as theactive ingredients, polysaccharides with a molecular weight of from10,000 to 50,000 and water soluble lignin with a molecular weight offrom 50,000 to 100,000 prepared by subjecting bagasse to the enzymeactivity and extracting it are the same as the data described in theaforesaid JPA Laid-Open No. S55-157517. There is not described datum ofany anti-virus test on the components extracted by boiling andextracting bagasse. (It should be noted that abstract description aboutthe boiling and extraction was deleted later in an invalidation appealprocedure.) Therefore, the contents of JPA Laid-Open No. S57-106624 aresubstantially the same as and nothing more than those of JPA Laid-OpenNo. S55-157517. As summarized above, basidiomycetes containing fungisuch as shiitake mushrooms and fomes japonicus are known to bephysiologically active and generally used in health food. Some of theextracts from these fungi cultured bagasse are known to have anti-viruseffects. To extract them, mycelia of basidiomycetes, fungi, or enzymesproduced thereby are necessary.

As already described above, when infectious diseases, especially thosecaused by bacteria, take place, antibiotics are generally administered.In these cases, especially, where the antibiotics are administered whenthe causal bacteria have proliferated above a certain level, all thebacteria die at once and endotoxin present in the bacteria moves to ahost, which may cause endotoxin shock in the host. Besides this suddenmove of the endotoxin in blood caused by the antibiotics, the bacteriaor the endotoxin thereof may circulate in blood to cause septicemia orsepticemia shock.

To prevent or remedy these diseases caused by endotoxin, someanti-endotoxin agents have been reported: a method of using an antibodyas an anti-endotoxin agent to remedy diseases caused by endotoxin (JPALaid-Open No. S61-500355, National Publication of Translation of PCTApplication No. H4-506447, JPA Laid-Open No. H2-104534, JPA Laid-OpenNo. H2-134329, JPA Laid-Open No. H6-62844, and National Publication ofTranslation of PCT application No. H6-501931), a method of usinghirudine which is a thrombin inhibitor (JPA Laid-Open No. H6-165691), amethod of using denatured C reactive protein (National Publication ofTranslation of PCT Application No. H7-501545), a method of using1,4-thiazine derivatives (JPA Laid-Open No. S63-301876), a method ofusing heterocyclic derivatives (JPA Laid-Open No. H3-240779), a methodof using an anti-endotoxin agent comprising taurine as an activeingredient (JPA Laid-Open No. H10-158158), a method of using a novelcompound (JPA Laid-Open No. H5-194470).

Recently, a vaccine adjuvants attract attention as an additive tovaccine because they are considered to play an important role forenhancing antigenicity of the vaccine. Particularly, the vaccineadjuvant is indispensable for an inactivated vaccine, because expressionof the effect of the vaccine is unstable.

Currently, adjuvants clinically used for man and animals are thosetopically used together with vaccines, for example, plant oils such assesame oil and colza oil, mineral oils such as complete Freund'sadjuvant and incomplete Freund's adjuvant, aluminum hydroxide, andaluminum sulfate.

Studies have been made on vaccine adjuvants. Usually, an adjuvant ismixed with vaccine and is injected or orally administered. Seeking for asafer and natural adjuvant effect, studies have been reported on orallyadministered adjuvants derived from natural products. As oral adjuvants,reported are an adjuvant for influenza virus vaccine containingShouseiryutou as an effective ingredient (JPA Laid-Open No. H7-173069),avian oral adjuvant containing effective ingredient NaF (JPA Laid-OpenNo. H10-59869), and an oral adjuvant containing an effective ingredientmutant enterotoxin (National Publication of Translation of PCTapplication No. H10-505059) were reported. As adjuvants derived fromplants, the following has been reported: a specific lipid emulsion typeof adjuvant containing a fatty oil originating from a plant,polysaccharide vaccine adjuvant comprising purified and detoxicatedendotoxin and trehalose dimicolate (JPA Laid-Open No. S63-22029), anadjuvant composition containing a synthetic hydrophobiclipopolysaccharide and a surfactant component originating from a plant(JPA Laid-Open No. H5-255117), vaccine containing asemannan extractedfrom aloe as an adjuvant (National Publication of Translation of PCTapplication No. H7-506565).

Also, use of detoxicated toxins such as mutated cholera toxin andmutated heat-labile toxin and cytokine IL-12 are studied (ExperimentalMedicine (Jikken Igaku), 17, 199(1999)).

In the livestock and fishing industries, it is desired to grow domesticanimals, fish and shrimps faster for shipping them, or to raiseproductivity by growing weak domestic animals, fish or shellfish whichare usually considered to be too small and too weak to grow to beshipped. Many studies have been made for the purpose of growing animalsfaster by increasing feed efficiency, of changing taste and flavor offeed to make inexpensive and less-preferred feed more effective, or ofgrowing weak domestic animals, fish and shellfish which are usuallyconsidered to be too weak to grow to be shipped.

As a means to increase a weight of a domestic animal, the following hasbeen reported: an additive for animal feed containing soy bean, toadvenom, araliaceae, and animal gallbladder (JPA Laid-Open No. H7-313070),a method of using a mixture of beer yeast and ethanol (JPA Laid-Open No.S48-61266), a method of using an antibiotic, multhiomycin (JPA Laid-OpenNo. S52-54013), a method of using a titanium complex (JPA Laid-Open No.S58-76050), a method of using globulin-containing substance (JPALaid-Open No. S61-132143), and a method of using a carbazate (JPALaid-Open No. S61-145156).

As a method for promoting animal growth, the following has beenreported: a method of using β-phenethanolamine (JPA Laid-Open No.S59-155343), a method of using an epithelial cell growth factor (JPALaid-Open No. S62-240625), a method of using a morphorin derivative (JPALaid-Open No. H1-6262), and a method of using forskolin (JPA Laid-OpenNo. H1-320956).

As a method for decreasing a feed demanding rate to thereby improve anefficiency of weight increase of domestic animals, the following hasbeen reported: a method of using fruit-origin vinegar (JPA Laid-Open No.S48-103364), a method of using a porcine prolactin (JPA Laid-Open No.H1-230531), and a method of using a product of a bacteriolytic enzymeand protease (JPA Laid-Open No. H2-207756).

As a method of changing preference for animal food to thereby increase aweight, a method of using hexanol or hexanal was reported (JPA Laid-OpenNo. H7-313067).

As a method of decreasing diseases such as diarrhea to thereby promotegrowth and weight increase, the following has been reported: a method ofusing a fructoligosaccharide (JPA Laid-Open No. S60-34134), a method ofusing an inulo-oligosaccharide (JPA Laid-Open No. S61-40754), a methodof using galactosyl disaccharide (JPA Laid-Open No. H4-360652),a methodof using a specific polysaccharide having β-1,3-glucan as a main chain(JPA Laid-Open No. H7-50999), an agent for increasing weight andenhancing immunity comprising, as an active ingredient, bacterial cellsdeprived of capsules (JPA Laid-Open No. H2-11519), and a method forenhancing immunity and increasing weight using a feed containingcommon-purslane (JPA Laid-Open No. H6-141784).

The preventive or remedial effect, the anti-endotoxin effect, and thevaccine adjuvant effect are all related to immunity. However, theirfunctional mechanism are different from one another, and, accordingly,all of the preventive or remedial agents cannot work as ananti-endotoxin agent or a vaccine adjuvant. The preventive or remedialeffect is that against viruses or bacteria causing infectious diseases,and is different from that against endotoxin produced by bacteria. Somehaving a greater preventive or remedial effect for infection raise anantibody titer of a vaccine, but others may cancel the effect of avaccine by attacking an attenuated vaccine when they coexist togetherwith the vaccine. The anti-endotoxin effect and the vaccine adjuvanteffect are different from each other both in an object to be affectedand in functional mechanism. As a consequence, a natural material havingall of these effects has not been reported.

Natural preventives or remedies and anti-endotoxin agents previouslyreported have limited uses, because they must be orally administered ina large amount in order to express effects, or they have so strongtaste, smell, or flavor that, when added to food or feed in theireffective amounts, they affect taste or smell of food or feed, or raisecosts. Therefore, a material is desired which has taste or smellallowable to be added to a wide range of food or feed, express apreventive or remedial effect in a small dosage against infectiousdiseases, and is inexpensive and of natural product-origin. Most of thenatural preventive or remedy agents and anti-endotoxin agents previouslyreported comprise a specific ingredient as an active ingredient and itis apprehended that a long term or high dosage of them causes sideeffects. Therefore, among natural materials which show a preventive orremedial effect for infectious diseases in a small dosage, particularlyone which comprises a plurality of active ingredients and is morenatural, is desired.

Vaccine adjuvants previously reported include chemical compoundadjuvants, inorganic adjuvants and biological adjuvants. Those arepurified compounds, inorganic materials, or detoxicated ones ofenterotoxin or endotoxin produced by bacteria. These conventionally usedadjuvants do not steadily express the effects and sometimes cause a sideeffect of producing IgE. Recently, a natural and safer adjuvant,particularly one expressing the effects via an oral administration, isdesired.

Growth promoters previously reported include chemical compounds, plantsor extracts therefrom, microorganisms such as yeasts, waste materialssuch as deoiled soy beans, and biologically active substances containedin enzymes, proteins, or cells. However, there were only a few growthpromoters which were inexpensive and easily prepared from a naturalsource.

An object of the present invention is to provide a preventive or remedyfor infection, a preventive or remedy for endotoxin shock (ananti-endotoxin agent), a vaccine adjuvant and a growth promoter, whichare safe and effective to man or animals.

Another object of the present invention is to provide a food or ananimal feed comprising the preventive or remedy for infection, theanti-endotoxin agent, the vaccine adjuvant or the growth promoter.

SUMMARY OF THE INVENTION

To attain aforesaid objects, the present inventors have made studies onfood safe to man or animals which food can be produced at low costs andhas a preventive or remedial effect for infection, an anti-endotoxineffect, a vaccine adjuvant effect or a growth promoting effect. As aresult, the inventors have found that an extract obtained by treatingsugar cane, which has been used as food from an ancient time, has thepreventive or remedial effect against infection caused by bacteria orviruses, the anti-endotoxin effect, the vaccine adjuvant effect and thegrowth promoting effect to make the present invention.

Thus, the present inventions are a preventive or remedy for infections,an anti-endotoxin agent, a vaccine adjuvant, and a growth promoter eachcomprising a sugar cane-derived extract as an active ingredient.

Particularly, the present inventions are the preventive or remedy forinfection, the anti-endotoxin agent, the vaccine adjuvant, and thegrowth promoter, each comprising, as an active ingredient, a fractionobtained by treating a raw material selected from the group consistingof sugar cane juice, a liquid extract from sugar cane, and sugarcane-derived molasses, in column chromatography with a fixed carrier.

The present inventions are the preventive or remedy for infection, theanti-endotoxin agent, the vaccine adjuvant, and the growth promoter,comprising, as an active ingredient, a sugar cane-derived extractobtained by extracting bagasse with water, a hydrophilic solvent or amixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an elution pattern obtained in column chromatographyperformed in Preparation Example 1.

FIG. 2 shows an elution pattern obtained in column chromatographyperformed in Preparation Example 2.

FIG. 3 shows absorbance, electric conductivity, and sugar composition ofthe fractions obtained by separation with an ion exchange resinperformed in Preparation Example 6.

FIG. 4 shows an elution pattern obtained in gel permeationchromatography on the extract of Preparation Example 3, performed inTest Example 4.

FIG. 5 shows an elution pattern obtained in gel permeationchromatography on the extract of Preparation Example 5, performed inTest Example 4.

FIG. 6 shows an elution pattern obtained in gel permeationchromatography on molecular weight markers, performed in Test Example 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the specification, the terms “a preventive or remedy for infection”mean an agent having an effect of preventing or remedying infection withbacteria or viruses. Such an effect includes a preventive or remedialeffect through control of immunological systems and that through othermechanisms.

In the specification, “an anti-endotoxin effect” includes a preventiveor remedial effect for diseases caused by endotoxin such as an effect ofdecreasing death by endotoxin shock or septicemia and even an effect fororal diseases by periodontal bacteria. Further, an effect as a radicalscavenger and an effect of suppressing inflammatory cytokine may beexpected.

In the specification, “a vaccine adjuvant function” or “a vaccineadjuvant effect” is an effect of enhancing functions of an antigen, thatis, an immune promoting effect to enhance an immune response.Specifically, it is the effect of increasing an antibody titer tothereby increase the effect of vaccination by administering the adjuvantin a specific period of time before or after vaccination.

In the specification, “a growth promoting effect” includes an effect ofpromoting growth of infantile man or animals and an effect of increasingbody weight of thin man or animals. In the specification, the growthpromoting effect is not distinguished from the weight increase-promotingeffect.

In the specification, the term “animals” means vertebrates other thanman, including mammals, birds and fish, for instance, domestic animalssuch as cows, pigs, and horses, poultry such as domestic fowls andquails, fish such as young yellowtails, sea breams, flatfish, swellfish,amberjack, sweetfish, eels, trout, carps, and goldfish, and companionanimals such as dogs and cats.

In the specification, a sugar cane-derived extract is an extractobtained from sugar cane as a raw material.

In one embodiment of the invention, the sugar cane-derived extract is afraction obtained by treating a raw material selected from the groupconsisting of sugar cane juice, extracted liquid from sugar cane andsugar cane-derived molasses (may be referred to simply as a raw materialhereinafter) in column chromatography with a fixed carrier. Morepreferably, the sugar cane-derived extract is a fraction obtained bypassing the raw material through a column packed with a syntheticadsorbent as the fixed carrier and eluting adsorbed substances on thesynthetic adsorbent with a solvent selected from the group consisting ofwater, methanol, ethanol, or a mixture thereof, or a fraction whichabsorbs light of a wave length of 420 nm out of fractions obtained bytreating the raw material in column chromatography utilizing differencesin affinity to an ion exchange resin packed in a column as the fixedcarrier. It is preferred to subject the fraction absorbing 420 nm lightto electrodialysis to thereby decrease or, more preferably, remove saltsin the fraction.

Color values of sugar cane-derived raw materials, intermediates, andproducts were previously evaluated absorbance at 420 nm. The absorbanceat 420 nm is slightly affected by a pH of a sample, so that the pH ofthe sample is adjusted to about neutral pH prior to the measurement ofabsorbance. In the invention, absorbance is measured after adjustingsamples' pH to a range of from 6 to 8. As will be described in theExamples, when 0.25 g of the freeze-dried powder of the obtainedfraction is dissolved in 0.5 mM phosphate buffer (pH 7.5) to a totalvolume of 100 ml and its absorbance is measured at 420 nm in a cell witha path length of 1 cm, fractions having an absorbance at 420 nm of 0.8or higher has have a higher effect of the present invention. However,the absorbance at 420 nm is a measure for content of color derived fromsugar cane and it is unknown whether the absorbance at 420 nm isattributable to the present active ingredients and, further, the contentof color in sugar cane vary depending on their production places andspices, so that there may not necessarily be proportional relationbetween the present effect and the absorbance at 420 nm among variousextracts from sugar canes of different production places and spices.Therefore, absorbances at 420 nm of a plurality of fractions obtainedfrom a raw material are measured and the fractions having a relativelyhigher absorbance are the fraction of the present invention.

When column chromatography is performed with a synthetic adsorbentpacked in a column, active ingredients are adsorbed to the syntheticadsorbent upon passing a raw material through the column, because theyhave very strong affinity to the adsorbent. Subsequently, the adsorbedingredients are desorbed and eluted with a solvent.

When an ion exchange resin is used as the fixed carrier, affinity of thepresent active ingredients to the resin is not so strong as adsorption.However, there is a difference of affinity to the ion exchange resinbetween the active ingredients and the other ingredients. Based on thedifference in an eluting speed, the active ingredients can be separatedfrom the other ingredients by feeding the raw material to the column andthen passing water as an eluent.

In another embodiment of the invention, a sugar cane-derived extract isan extract obtained by extracting sugar cane-derived bagasse with aliquid selected from water, hydrophilic solvents, and a mixture thereof,more preferably, an extract obtained by extracting bagasse, which is aresidue after milling sugar cane for milling juice with a liquidselected from water, hydrophilic solvents, and a mixture thereof.

In the invention, sugar cane juice includes mill juice obtained bymilling sugar cane, extracted juice obtained by extracting sugar canewith water, clarified juice obtained by treating with lime in a sugarmill, and concentrated juice.

In the invention, a liquid extract of sugar cane includes an aqueoussolution obtained by extracting sugar cane with a widely used organicsolvent, concentrating, drying and re-dissolving in water. Examples ofthe organic solvent include alcohols such as methanol, ethanol and acombination thereof. A mixture of the alcohol with water may be used.

In the invention, sugar cane-derived molasses includes molasses obtainedby centrifuging a mixture of sugar crystals and a mother liquor obtainedin a crystallization process and separating molasses from the sugarcrystals, such as first molasses, second molasses, final molasses in asugar mill, and affination syrup, first to seventh molasses , andrefinery final molasses in a sugar refinery. Also use is made of aresidue deprived of saccharides such as an isolated liquor obtained inalcoholic fermentation of these molasses as a raw material.

In the invention, bagasse typically means bagasse exhausted in sugarmanufacturing processes in a sugar mill. Here, the bagasse exhausted insugar manufacturing processes in a sugar mill include not only a finalbagasse from a final press but also finely crushed sugar cane which isbitten to be in presses of from the first press to the final press.Preferably, bagasse exhausted after milled for mill juice in the millingprocess in the raw sugar plant is used. The waste bagasse from themilling process varies in a moisture content, sugar content andcomposition depending on sugar cane species, and its harvest time.However, in the present invention, any bagasse can be used. Use is madesimilarly of bagasse exhausted from non-centrifugal sugar (KOKUTOU)plant, which remains after milling sugar cane. In a small scale ofpractice in a laboratory level, use may be made of bagasse remainingafter squeezing sugar cane for press juice.

More specifically, the sugar cane-derived extract can be prepared asfollows.

First, the present method of column chromatographic treatment will beexplained.

Sugar cane juice, liquid extract of sugar cane obtained by extractionwith a solvent or sugar cane-derived molasses (hereinafter simplyreferred to as a raw material) is passed through a column packed with afixed carrier. The aforesaid raw material can be used as such or afterdiluted with water to a desired concentration. It is preferred to filterthe raw material before treated with the column to remove any foreignsubstances. The filtration means is not restricted to particular one anduse may be preferably made of various means widely used in the foodindustry such as screen filtration, diatomaceous earth filtration,precision filtration and ultrafiltration.

As the fixed carrier, a synthetic adsorbent or an ion exchange resin ispreferred.

First, a preferred embodiment will be explained where a syntheticadsorbent is used as the fixed carrier. As the synthetic adsorbent, usemay be preferably made of organic resins such as aromatic resins,acrylic acid type methacrylic resins and acrylonitrile aliphatic resins.More preferred are the aromatic resins, particularly unsubstitutedaromatic resins. As the synthetic adsorbent, aromatic resins, forexample, styrene-divinylbenzene resin may be used. As the aromaticresin, use may be made of porous resins, for example, aromatic resinshaving hydrophobic substituents, unsubstituted aromatic resins andporous resins such as aromatic resins obtained by subjectingunsubstituted type aromatic resins to a special treatment. Morepreferably, use may be made of the aromatic resins obtained bysubjecting the unsubstituted type aromatic resins to a specialtreatment. These synthetic adsorbents are commercially available as, forexample, Diaion® series, such as HP-10, HP-20, HP-21, HP-30, HP-40 andHP-50 (trade names, ex Mitsubishi Chemicals Inc.: these areunsubstituted aromatic resins), SP-825, SP-800, SP-850 and SP-875, SP-70and SP700 (trade names, ex Mitsubishi Chemicals Inc.: these are aromaticresins obtained by subjecting the unsubstituted type aromatic resins toa special treatment); SP-900 (trade name, ex Mitsubishi Chemicals Inc.,aromatic resin), Amberlight® series such as XAD-2, XAD-4, XAD-16 andXAD-2000 (trade names, ex Organo Inc.: these are aromatic resins);Diaion® series SP-205, SP-206 and SP-207 (trade names, ex MitsubishiChemicals Inc.: these are aromatic resins having hydrophobicsubstituents), HP-2MG and EX-0021 (trade names, ex Mitsubishi ChemicalsInc.: these are aromatic resins having hydrophobic substituents),Amberlight® series XAD-7 and XAD-8 (trade names, ex Organo Inc.: theseare acrylic ester resins), Diaion® series HP1MG and HP2MG (trade names,ex Mitsubishi Chemicals Inc.: these are acrylic acid type methacrylicresins), Sephadex® series LH20 and LH60 (trade names, ex PharmaciaBiotech Inc.: these are cross-linked dextran derivatives) and the like.Among these, SP-850 is particularly preferable.

The amount of the fixed carrier varies depending upon a size of thecolumn, a type of a solvent and a type of the fixed carrier. Apreferable amount is 0.01 to 5 times, as a wet volume, as large as asolid content of the raw material.

Upon passing the raw material through the aforesaid column, ingredientshaving the present effect in the raw material are adsorbed to thecarrier, and most parts of sucrose, glucose, fructose and inorganicsalts pass through the column.

The ingredients adsorbed to the fixed carrier are eluted with a solvent.In order to efficiently elute the ingredients having the present effect,it is preferred to wash the column sufficiently with water to removeremaining sucrose, glucose, fructose and inorganic salts out of thecolumn before the elution, whereby the adsorbed ingredients havingintended effect are recovered efficiently. The eluting solvent isselected from water, methanol, ethanol and a mixture thereof. Preferenceis given to a mixed solvent of water with alcohol, particularly anethanol-water mixture. A mixture of ethanol and water in a volume ratioof 50/50 to 60/40 is more preferred, because the ingredients havingintended effects are eluted efficiently at room temperature. Byelevating a column temperature, one can elute the intended ingredientshaving the present effect with a lower ethanol ratio in theethanol-water mixture which can out. Here, a pressure in the column isan atmospheric pressure or higher. The ingredient having the presenteffect are present in the fractions eluted with the aforesaid solvent.An elution rate varies depending upon a column size, a solvent type anda type of the fixed carrier, and is not restricted to a particular rate.However, SV is preferably in the range of 0.1 to 10 hours⁻¹, where SV isa space velocity representing how many times a liquid volume as theresin volume is passed per hour.

The ingredients having the present effects may be obtained preferably inthe following manner, but is not limited to it. That is, a raw materialis passed through a column packed with an unsubstituted aromatic resinhaving a wet volume 0.01 to 5 times as much as a solid content of theraw material at a column temperature of 60 to 97° C. After washing theresin in the column with water, the ingredients adsorbed to the resinare eluted at a column temperature of 20 to 40° C. with a mixture ofethanol and water in a volume ratio of 50/50 to 60/40 and the fractionsare collected until a volume of the eluent collected from the beginningof the elution becomes 4 times as much as the wet volume of theaforesaid resin.

Meanwhile, a preferred embodiment with an ion exchange resin as thefixed carrier will be described below. Ion exchange resins areclassified into a cation exchange resin and an anion exchange resin froma viewpoint of an ion exchanging property. In the present invention, acation exchange resin is preferably used. More preferably, a stronglyacidic type, sodium ion form, or potassium ion form of cation exchangeresins are used. Ion exchange resins are also classified, from amorphological viewpoint, into a gel type resin and a porous type resinsuch as a porous type, a macroporous type, and a highly porous type. Inthe present invention, a gel type ion exchange resin is preferably used.More preferably, a gel type cation exchange resin of a strongly acidictype, in a sodium ion form or in potassium ion form is used. Such ionexchange resins are commercially available, for example, include Diaion®series SK1B, SK104, SK110, SK112, SK116 (all trade names, ex MitsubishiChemicals Co.), UBK530, UBK550 (chromatographic grades, trade names, exMitsubishi Chemicals Co.), Amberlite® series Amberlite IR120B, IR120BN,IR124, XT1006, IR118, Amberlist 31, chromatographic grade AmberliteCG120, CG6000 (trade names, ex Organo Co.), Dowex® series such as HCR-S,HCR-W2, HGR-W2, Monosphere 650C, Marason C600, 50Wx2, 50Wx4, 50Wx8 (alltrade names, ex Dow Chemical Japan Co.), Muromac 50WX (trade name, exMuromachi Chemical Industry Co.), and Purolite series C-100E, C-100,C-100x10, C-120E, PCR433, PCR563K, PCR822, PCR833, PCR866, PCR883,PCR892,PCR945 (all trade names, ex AMP Ionex Co.). Among these, UBKseries are particularly preferred.

The amount of the fixed carrier varies depending upon a size of columnand a type of the fixed carrier. Preferably, the amount, as a wetvolume, is 2 to 10,000 times, more preferably 5 to 500 times, as largeas a solid content of the raw material.

The raw material is passed through the aforesaid column and thensubjected to a chromatographic treatment with water as the eluent. Outof many fractions obtained, those absorbing 420 nm light are collectedto obtain the intended extract. Hereinafter, this treatment method maybe referred to as ion chromatographic separation.

The separation conditions vary depending on composition of the rawmaterial and a type of the fixed carrier. In a single column batchseparation using degassed water as the eluent, preferred conditions areas follows: a flow rate, as SV, of from 0.3 to 1.0 hr−1, charge amountof the raw material of from 1 to 20% of a volume of the ion exchangeresin, and temperature of from 40 to 70° C. For-each of the fractionsobtained by this separation method, absorbance at a wave length of 420nm, electric conductivity, which is a measure of a salt content,contents of sucrose, glucose, and fructose are determined. When thesedata are plotted against time, peaks in the absorbance at 420 nm, in theelectric conductivity, in the sucrose content and in the content of areducing sugar are found in this sequence. In FIG. 3, the fractions offrom 3rd to 14th are collected as the fractions absorbing the light of awave length of 420 nm. Particularly, fractions of from 3rd to 8th arepreferred. The entire less-sugar fractions consisting of fractions of1st to 9th, including fractions 3rd to 8th, and the fractions of 18th to30th, may as well be used as the present extract, though a concentrationof the active ingredients is lower. For a pseudo moving-bed continuousseparation method, general separation conditions cannot be presentedhere because a charge rate of the raw material, a flow rate of aneluent, and a withdrawal flow rate are set according to a composition ofthe raw material, a type of the fixed carrier, and an amount of ionexchange resin.

The present fractions obtained by pseudo moving-bed continuousseparation using a 2nd molasses from a raw sugar mill as the rawmaterial comprise, at most 6% of sucrose and at most 90% of non-sugarcomponent, based on a solid content, and have an apparent purity of 10%,though the composition varies depending on a type of the raw materialand a separation capability of the ion exchange resin. The apparentpurity is a percentage of a polarization per solid content (Brix:Bx),where polarization is an angle of rotation measured with a sucrometerrelative to a pure sucrose standard of a specified concentration.

The present fractions obtained by a single column batch separationmethod using a 2nd molasses as the raw material contain about 5% ofpolyphenols, about 44.7% of electrically conductive salts and about 5%of sugar, based on a freeze-dried solid content.

It is clear that the present active ingredients are contained more inthe fractions corresponding to the peak of absorbance at a wave lengthof 420 nm, but it has not yet been clear whether the active ingredientsthemselves absorb light of 420 nm.

The aforesaid fractions absorbing light at a wave length of 420 nm orthe less-sugar fractions may be treated further by electrodialysis tothereby decrease or remove salts contained in the fractions. Thefractions obtained by column chromatography with an ion exchange resincontain salts in an amount so much as about 40% of sulfate ash, based ona dried solid. Consequently, the fractions taste very salty and affecttaste of foods. To allow man to take the fractions, the salt contentshould be decreased because too much intake of salts is bad for health.This applies also to animals and there is a maximum permissible saltintake. Especially for domestic animals, an amount of each kind of saltto be given is regulated, to which salt contents in a formula feed isconformed. Therefore, for application in domestic animal foods, thesugar cane-derived extract preferably contains a lower amount of thesalts. Accordingly, it is preferred to decrease the salts contents ofthe obtained fractions.

In desalinization by electrodialysis, cations such as sodium ion,potassium ion, calcium ion, and magnesium ion are removed almost equallyregardless of ion species. As to anions, it is known that chloride ionis selectively removed than sulfate ion which is not removed so well.Cation and anion are removed in an equivalent ratios.

Now, a method of preparing the sugar-cane derived extract by extractingbagasse will be explained. The bagasse extract is prepared by extractingbagasse with a solvent selected from the group consisting of water,hydrophilic solvents and a mixture thereof. Examples of the hydrophilicsolvents include lower alcohols such as methanol and ethanol, ketonessuch as acetone, and acetates such as methyl acetate and ethyl acetate.Ethanol is a preferred hydrophilic solvent. A preferred solvent for theextraction is a mixture of ethanol and water in a volume ratio of atmost 60/40, more preferably at most 50/50. For efficient extraction, anextraction temperature of from 50 to 100° C. is preferred. An extractiontime is usually 1 to 3 hours, though this varies depending on bagasse'ssource, type, and state. Any commonly used method for extraction maybeused such as extraction in a container where bagasse and the extractingsolvent are placed together, extraction by circulating the extractingsolvent, continuous extraction using, for example, a Desmet extrator anda Lurgi extractor. The extract from the bagasse contains muchsaccharides, so that it may be subjected to column chromatograpictreatment similar to the aforesaid method to thereby remove thesaccharides.

The present active ingredients may be obtained by condensing the extractfrom cane sugar prepared by any of the aforesaid various methods, in aconventional method such as evaporation of the solvent under vacuum andfreeze-drying. The active ingredients thus obtained may be stored in aform of a condensed liquid with at least 20% of solid content or powder.Preferably, it is stored in a refrigerator, particularly when it isliquid.

As will be shown later in Test Example 4, the present sugar cane-derivedextract comprises not only one active ingredient but a plurality ofactive ingredients and has different condensed compositions of theactive ingredients depending on the method of preparation.

Preventives or remedies for infection hitherto known generally comprisea single active component or a plurality of similar active components,so that it is apprehended that a long-term or large amount of dosagewould cause side effects. In contrast, the present sugar cane-derivedextract comprises many components of a wide range of molecular weightsand is more natural.

The present sugar cane-derived extract shows preventive or remedialeffects for infection against bacteria and viruses in an animalexperiment where the sugar cane-derived extract was orally administeredto mice (see Examples 1 to 4). It is believed that the present sugarcane-derived extract control immunological system to thereby prevent orremedy infection.

Therefore, the present invention may be applied to prevent or remedydiseases caused by weakness or deficiency of immunological functionthrough control of immunological function of man or animals. The presentinvention may be applied also to prevent or remedy various kinds ofinfectious disease.

Such diseases are not limited to particular ones. In the case of man,examples include articular rheumatism, glomerulonepheritis, hemolyticanemia, bronchial asthma, Behcet's disease, Hashimoto's disease,polymyositis, systemic lupus erythematosus, autoimmune diseases such asprogressive systemic sclerosis and some sorts of tumors, infectiousdisease of a whole body, respiratory systems, urinary tracts,intestines, intra-abdomens, mucus membranes, or circulatory organs,various kinds of infectious diseases of children with nutritionaldisturbances, aged persons, or those who are under administration ofanticancer agents or operative invasions. In the case of animals,examples include diarrhea, epidemic pneumonia and infectiousgastroenteritis of pig, avian pneumonia and Marek disease, bovinediarrhea, pneumonia and mastitis, and feline immnodeficiency syndromeand leukemia. Also, infectious diseases of cultured fish are not limitedto particular ones and examples include bacterial infection such asstreptococcosis, pasteurellosis and virus infection.

Examples of bacterial infection include human Salmonellosis (Salmonellaenteritidis, S. dublin), Vibrio parahaemolyticus, typhoid fever(Salmonella typhi), infectious E. coli infectious disease (Escherichiacoli), tuberculosis (Mycobacterium tuberculosis), bacillary dysentery(Shigella dysenteriae, S. flexneri), pertussis (Bordetella pertussis),diphtheria (Corynebacterium diphtheriae), Hansen disease (Mycobacteriumleprae), plague (Yersinia pestis), bovine mastitis (Staphylococcusaureus, Klebsiella pneumoniae, Streptcoccus agalactiae, Actinomycespyogenes), Brucella disease (Brucella abortus), Campylobacter disease(Campylobacter fetus), anthrax (Bacillus anthracis), Johne's disease(Mycobacterium avium), bovine infectious keratoconjuctivitis (Moraxellabovis), pasteurellosis (Pasteurella multocida and Pasteurellahaemolytica), trichophytia interdigitalis (Fusobacterium necrophorum),glanders (Bordetella mallei), horse infectious uteritis (Taylorellaequigenitalis), relapsing fever (Borrelia theileri), porcine atrophicrhinitis (Bordetella bronchiseptica), porcine erysipelas (Erysipelothrixrhusiopathiae), Glasser disease (Haemophilus parasuis), chicken diarrheawith white stool (Salmonella pullorum), domestic avian cholera(Pasteurella multocida), infectious coryza (Haemophilus paragallinarum),atypical mycobacterial disease (Mycobacterium avium), canine ocularleptospirosis (Leptospira canicola), tetanus (Clostridium tetani),ichthyic Enterococcus infection (Enterococcus seriolicida),pasteurellosis (Pasteurella piscicida), Vivrio disease (Vivrioanguillarum), Edwardsiella infection (Edwardsiella tarda), coldwaterdisease (Flavobacterium psychrophilus), red mouth disease (Yersiniaruckeri), Aeromonas infection (Aeromonas hydrophila), nocardiosis(Nocardia asteroides, Nocardia seriolae).

Examples of viral diseases include human influenza (Humaninfluenzavirus), human herpes (Human herpesvirus 3), humanimmunodeficiency syndrome (Human immunodeficiency syndrome virus),poliomyelitis (Polio virus), rubella (Rubella virus), measles (Measlesvirus), variola (Variola virus, Japanese encephalitis (Japaneseencephalitis virus), epidemic parotitis (Mumps virus), Ebola hemorrhagicfever (Ebola virus), dengue fever (Dengue virus), Marburg disease(Marburg virus), Lymphocytic choriomeningitis (Lymphocyticchoriomeningitis virus), human T-lymphocyte leukemia (HumanT-lymphotrophic virus), Yellow fever (Yellow fever virus), Bovineinfectious rhinotracheitis (Bovine herpesvirus 1), bovine Foot-and-mouthdisease (Foot-and-mouth disease virus), bovine ephemeral fever (Bovineephemeral fever virus), Cowpox (Cowpox virus), Akaban disease (Akabanevirus), Ibaraki disease (Ibaraki virus), Bluetongue (Bluetongue virus),Shipping fever (Bovine parainfluenza virus), Rift Valley fever (RiftValley fever virus), equine infectious anemia (Equine infectious anemiavirus), equine arteritis (Equine arteritis virus), Borna disease (Bornavirus), equine rhinopneumonitis (Equid herpesvirus 4), easter equineencephalitis (Eastern equine encephalitis virus), porcine transmissiblegastroenteritis (Porcine transmissible gastroenteritis virus), porcinereproductive and respiratory syndrome (Porcine reproductive andrespiratory syndrome virus), Aujeszky's disease (Pseudorabies virus),hog cholera (Hog cholera virus), porcine vesicular disease (Swinevesicular disease virus), swine inclusion body rhinitis (Suidherpesvirus 2), avian infectious bursal disease (Infectious bursaldisease virus), Newcastle disease (Newcastle disease virus), avian pox(Fowlpox virus), Marek's disease (Marek's disease virus), Infectiouslaryngotracheitis (Infectious laryngotracheitis virus), Avian infectiousbronchitis (Avian infectious bronchitis virus), canine rabies (Rabiesvirus), canine distemper (Canine distemper virus), infectious hepatitis(Canine adenovirus 1), canine parvovirus infection (Canine parvovirus),Feline leukemia (Feline leukemia virus), Feline immnodeficiency syndrome(Feline immnodeficiency virus, feline infectious peritonitis (Felineinfectious peritonitis virus), Feline panleukopenia (Felinepanleukopenia virus), ichthyic Iridovirus infection (Flouder virus),Infectious haemotopoietic necrosis (Infectious haemotopoietic necrosisvirus), infectious pancreatic necrosis (Infectious pancreatic necrosisvirus), fugu white mouth disease (unidentified).

Examples of fungous diseases include human coccidioidomycosis(Coccidioides immitis), histoplasmosis (Histoplasma capsulatum),bovinemastitis (Candida tropicalis), miscarriage (Aspergillusfumigatus), dermatomycosis (Trichophyton verrucosum), mucormycosis(Mucor rasemosus), equine gluttural pouch myosis (Aspergillus nidulans),infectious lymphadenosis (Histoplasma farciminosum), equinetrichophytosis (Trichophyton equinum), avian ingluviitis (Candidaalbicans), Aspergillus pneumonia (Aspergillus fumigatus), canineblastomycosis (Blastomyces dermatitidis), dermatomycosis (Microsporumcanis), lymphadenosis (Histoplasma capsulatum), Malasseziasis(Malassezia pachydermatis), ichthyic Saprolegnia infection (Saprolegniaparasitica), visceral mycosis (Saprolegnia diclina), mycetogenicgranuloma (Aphanomyces piscicida), Pythium infection (Pythium gracile),tympania (Candida sake).

Examples of infectious disease by mycoplasma include bovine Mycoplasmapneumoniae (Mycoplasma mycoides), Mycoplasma mastitis (Mycoplasmabovis), ovine infectious agalactia (Mycoplasma agalactiae), hog epidemicpneumonia (Mycoplasma hyopneumoniae), avian chronic upper air passageinflammation (Mycoplasma gallisepticum).

Examples of infectious disease by rickettsia include human typhus(Rickettsia prowazekii), Q fever (Coxiella burnetii), feline scratchdisease (Bartonella henselae), bovine haemorrhagic fever (Ehrlichiaondiri), equine Potomac fever (Ehrlichia risticii), canine ehrlichiainfection (Ehrlichia canis).

Examples of infectious disease by chlamydia include human psittacosis(Chlamidia psittaci), bovine epidemic miscarriage (Chlamydia psittaci),sporadic bovine encephalomyelitis (Chlamydia pecorum), ovine epidemichydrohymenitis (Chlamydia pecorum), chlamydia hamarthritis (Chlamydiapecorum), feline chlamydia pneumonia (Chlamydia psittaci).

The present sugar cane-derived extract showed a significant increase ina survival ratio in an endotoxin model where the sugar cane-derivedextract was orally administered to experimental animals one day beforeand 6 hours after an intravenous administration of endotoxin. Thisindicates that the sugar cane-derived extract itself or its metaboliteacts on the endotoxin present in blood to decompose, agglomerate, orcause any change in a state of the endotoxin to neutralize it, tosuppress excessive activation of complement by the endotoxin, serves asa radical scavenger, suppress inflammatory cytokine, or decreasesefficiency of endotoxin through any mechanism, to thereby haveanti-endotoxin effects. Therefore, the present sugar cane-derivedextract can be used to prevent or remedy diseases caused by endotoxin.Such diseases are not limited to particular ones and include sepsiscausing severe general symptoms such as fever, chill, vomiting anddisturbance of consciousness, endotoxin shock, and oral diseases byendotoxin such as periodontal bacteria. Examples of bacterium havingsuch endotoxin include Gram negative baterium such as Escherichia coli(E. coli), pneumobacilli, proteus, pseudomonas aeruginosa, andenterobacter.

The present sugar cane-derived extract also acts as a vaccine adjuvantand a growth promoter, as will be demonstrated in the Examples.

Administration timing of the present preventive or remedy for infection,anti-endotoxin agent, or growth promoter is not limited. Theadministration timing of the present vaccine adjuvant is not limited andmay be before, on or after the day of administration of vaccine.Generally, it is administered on and/or after vaccination. Byadministrating before the vaccination, more reliable effects can beexpected. At least one administration is enough and continuous orintermittent administration over a period of from one day to one weekbefore the vaccination and from one week to two weeks after thevaccination is preferred. Further continued administration for 1 to 6months may be made with no problem.

A Dosage of the present preventive or remedy for infection,anti-endotoxin agent, vaccine adjuvants or growth promoter is notlimited and may be decided depending on purity and a form of the sugarcane-derived extract, type, health state or a stage of growth of anobject animal. For example, a dosage of the sugar cane-derived extractpowder prepared in Preparation Examples 1 to 7 described later in thespecification is 1 to 1000 mg, preferably 50 to 1000 mg per kg bodyweight.

The present sugar cane-derived extract may be administered in any meanssuch as oral, intravenous, intramuscular, subcutaneous, intracutaneous,intra-abdominal, intrarectal and hypoglossal admistraion, endermism,instillation to exert a preventive or remedial effect, anti-endotoxineffect, vaccine adjuvant effect, or growth promoting effect.

The present extract may be administered in any form. The extract in theform of liquid or powder may be administered as such or may be made intosolid or liquid preparation with a carrier for preparation by a knownmethod. Alternatively, the present extract, either prepared or notprepared, may be mixed in a food, a feed, or drinking water.

A solid preparation for oral administration may be made by addingdiluent bases, binders, bonding agents, disintegrator, lubricants andbrightener, colorants, flavor and odor controller, antioxidants, anddissolution aids to the present extract and making the mixture intopellets, coated pellets, granules, powder, or capsulated drugs.

The examples of the aforesaid diluent bases include starch, corn starch,dextrin, flour, wheat middling, bran, rice bran, rice bran oil cake,soybean cake, soybean powder, soybean oil cake, soybean flour, glucose,lactose, white sugar, maltose, plant oil, animal oil, hardened oil,saturated higher fatty acids, other kinds of fatty acids, yeast,mannitol, crystalline cellulose, silicon dioxide, silicic anhydride,calcium silicate, silicic acid, calcium hydrogenphosphate, calciumphosphate, and calcium dihydrogenphosphate.

Examples of the binders include polyvinylpyrrolidone, ethyl cellulose,methyl cellulose, gum arabic, tragacanth gum, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, sodium alginate, sodium casein, sodiumcarboxymethyl cellulose, propylene glycol, and poly(sodium acrylate).

Examples of the lubricant and brightener include magnesium stearate,talc and stearic acid.

As the colorant and flavor or essence, any such agents allowed to beadded to drugs, foods, or feeds may be used.

Examples of the antioxidants include ascorbic acid, α-tocopherrol,ethoxyqin, dibutylhydroxytoluene, butylhydroxyanisole and those allowedto be added to drugs, foods, or feeds. Tablets or granules may be coatedas desired.

An injection medicine may be prepared by adding pH adjusting agents,buffers, suspending agents, dissolving aids, stabilizers, isotonicagents, antioxidants, preservatives and processing them by a knownmethod. Here, the medicine may be freeze-dried medicine. Injection maybe made intravenously, subcutaneously, or intramuscularly.

Examples of the suspending agents include methyl cellulose, polysolvate80, hydroxyethyl cellulose, gum arabic, tragacanth gum powder, sodiumcarboxymethyl cellulose, and polyoxyethylenesorbitan monolaurate.

Examples of the dissolving aids include polyoxyethylene hardened castoroil, polysolvate 80, nicotinic amide and polyoxyethylenesorbitanmonolaurate.

Examples of the preservatives include methyl paraoxybezoate, ethylparaoxybezoate, and sorbic acid.

The present invention also relates to a food or an animal feedcomprising the aforesaid preventive or remedy for infection,anti-endotoxin agent, vaccine adjuvant or growth promoter. The food oranimal feed may be in a form of solid or liquid. Examples of the foodinclude confectionery, soft drinks, functional seasoning, and healthfoods. Examples of the animal feed include pet foods such as dog foodsand cat foods, domestic animal feeds and feeds for cultured fish andshellfish.

EXAMPLES

The present invention will be more specifically explained. Thedescription on a dose such as “10 mg/kg” or “10 mg/kg weight” means 10mg of a dose per kg of body weight.

Preparation Example 1

Six hundred and fifty liters of a mill juice (solid content of 18.8%)obtained in a sugar preparation process in a sugar mill were heated to80° C. with a juice heater and then filtered through a tubular typeultrafiltration membrane (Daicel Chemical Industries Ltd., type MH-25,an effective membrane area of 2 m²×3 tubes, exclusion molecular weightof 100,000) to obtain about 600 liters of the treated juice.

Fifteen liters of a synthetic adsorbent (SP-850, trade name, exMitsubishi Chemical Co.) were packed in a column provided with a waterjacket (column size: an inner diameter of 17.0 cm and a height of 100cm). The aforesaid treated juice was passed through the column at a flowrate of 30 liters/hour (Space Velocity=2 hours⁻¹). During the passage ofthe filtered sugar cane juice, water at 65° C. was always circulated inthe water jacket. Then, the column was washed by passing 45 liters ofion exchanged water through the column at a flow rate of 30 liters/hour(SV=2 hour⁻¹). After the washing with the ion exchanged water, it wasconfirmed that Brix of the effluent was about zero, measured by aHandref Brix meter (ex Atago Company, type N-1E). Then, an aqueous 55%ethanol solution (ethanol/water=55/45 in volume ratio) as an elutingsolvent was passed through the column at a flow rate of 30 liters/hour(SV=2.0 hour⁻¹) to elute ingredients adsorbed to the syntheticadsorbent. During the passage of the eluting solvent, water at 25° C.was always circulated in the water jacket. The effluent from the columnwas collected in each portion of 5 liters. The elution pattern is asshown in FIG. 1, where (1) indicates a starting point of passing thefiltered sugar cane juice; (2), a starting point of the washing with ionexchanged water; and (3), a starting point of the elution with theaqueous 55% ethanol solution. In the figure, the filled circles showabsorbance at 420 nm and the empty squares show Brix of each fraction.The fraction eluded with the aqueous 55% ethanol solution( correspondingto part “A” in FIG. 1) was condensed approximately 20 times under vacuumwith a concentrator. After freeze-drying the concentrate overnight, 460g of brown powder, i.e., sugar cane-derived extract, was obtained.

Preparation Example 2

Six hundred liters of a clarified juice (solid content of 11.7%)obtained in a sugar preparation process in a sugar mill were treated inthe same manner as in Preparation Example 1 except that the juice wasnot treated by ultrafiltration. The elution pattern is as shown in FIG.2, where (1) indicates a starting point of passing the clarified juice;(2), a starting point of the washing with ion exchanged water; (3), astarting point of the elution with the aqueous 55% ethanol solution. Thefraction “B” in FIG. 2, was collected and condensed under vacuum. Afterfreeze-drying the concentrate overnight, 225 g of brown powder, i.e.,sugar cane-derived extract, was obtained.

Preparation Example 3

One kilogram of dried bagasse obtained in a sugar mill was put in a bagmade of nylon net and the bag containing bagasse was placed in a tank,to which 25 liters of water at 80° C. was added, and extraction wascarried out with stirring. After one hour extraction, the liquid extractobtained was filtered with a cotton filter to remove foreign substances.The filtrate was concentrated under vacuum with a centrifugal thin-layerconcentrator. After freeze-drying the concentrate overnight, 26.31 g ofdark brown powder, i.e., sugar cane-derived extract, was obtained.

Preparation Example 4

Three hundred and fifty grams of dried bagasse obtained in a sugar millwere put in a nylon net bag and placed in a tank, to which 5250 ml of anethanol/water of a volume ratio of 50/50 was added, and extraction wascarried out with stirring. After two-hour extraction, the liquidobtained was filtered with a No. 2 filter paper, ex Advantec Toyo Co.,to remove foreign substances. The filtrate was concentrated under vacuumwith an evaporator. After freeze-drying the concentrate overnight, 6.72g of dark brown powder, i.e., sugar cane-derived extract, was obtained.

Preparation Example 5 Separation by Pseudo Moving-Bed ColumnChromatography Using an Ion Exchange Resin

A second molasses was subjected to ion exchange column chromatographicseparation using a pseudo moving-bed of a cation exchange resin, whichmolasses had been obtained by collecting sucrose crystals twice in aboiling pan in a sugar mill and centrifuging a remaining syrup to removecrystals.

The processes from raw material preparation to ion exchange columnchromatographic separation were run continuously, and the solid contentand composition shown below are those measured in a steady operation,although they vary a little with time.

The second molasses had a Brix of about 85. This concentration was toohigh to be treated by column chromatography, so that the molasses wasdiluted to a Brix of about 50. To this, slaked lime and sodium carbonatewere added to agglomerate impurities and then filtered throughdiatomaceous earth. The filtrate obtained had a Brix of 47.3,polarization of 23.6, purity of 49.9 and reducing sugar-content of 2.5%.This filtrate was subjected to ion exchange chromatography.

Ion exchange chromatography was carried out in a pseudo moving bedcontinuous separation method using a cation exchange resin, UBK530, exMitsubishi Chemical Co. The separation column packed with the resin had8 sections, each of which contained 6.5 m³ of the resin. The liquid feedand water as an eluent were continuously supplied to a different sectionat every predetermined time interval and a fraction containing sucroseand one containing less sucrose were taken out from different sectionsat every predetermined time interval. Operating conditions in a steadystate were as follows: the flow rate of the feed stream was 3 m³/hr; theeluent water flow rate was 13.5 m³/hr; the withdrawing rate of thefraction with less sucrose was 12.13 m³/hr; the withdrawing rate of thesucrose-containing fraction was 4.37 m³/hr; and the switching intervalwas 267 seconds. By this chromatographic treatment, thesucrose-containing fraction and the fraction with less sucrose wereseparated from each other. The former corresponds to fractions 10 to 17in FIG. 3, and the latter to fractions 1 to 9 and fractions 18 to 30.The sucrose-containing fraction had a sucrose content, determined byHPLC, of about 87% per solid weight and a Brix of about 35. Thisfraction was combined with the clarified juice and recycled to mainprocess to recover sucrose again. The fraction with less sucrose had asucrose content, determined by HPLC, of about 0.3% and a Brix of about8. This fraction was condensed in a vacuum pan to a Brix of 40.0,polarization of 2.3, purity of 5.8 and reducing sugar content of 5.4%.This fraction was freeze-dried overnight to be used in further tests.Zero point twenty five gram of the freeze-dried powder obtained wasdissolved in 0.5 mM phosphate buffer at pH of 7.5 a total volume of 100ml, of which absorbance at 420 nm was determined to be 1.11.

Prepation Example 6 Fractionation of a Second Molasses by Single ColumnBatch Separation Using an Ion Exchange Resin

A liquid prepared by treating a second molasses obtained in a sugar millwas subjected to single column batch ion chromatographic separation.

The liquid was prepared by diluting a second molasses and washing withsodium carbonate, and filtering through diatomaseous earth. This liquidraw material had a Brix of 47.4, polarization of 23.2, purity of 48.9and a reducing sugar content of 3.2%.

This liquid raw material was subjected to fractionation by single columnbatch ion chromatographic separation using FPLC system (Pharmacia Co.).The column was packed with 500 ml of gel type of a strongly acidiccation exchange resin in sodium form, UBK 530, ex Mitsubishi ChemicalCo. The column had an inner diameter of 26 mm and a height of 1000 mm,equipped with a flow adapter. Degassed distilled water as an eluent waspassed at a flow rate of SV=0.5 hr⁻¹ (4.17 ml/min) at 60° C.

About 25 ml of the raw material was fed to the column. Collection of theeffluent was started about 30 minutes after feeding the raw material.The effluent was collected for 3.6 min. per test tube (about 15 ml pertube), and the effluent of 30 tubes in total was recovered.

Each of the 30 fractions was analyzed for absorbance at 420 nm, electricconductivity, and sugar content. The results are as shown in FIG. 3. Tomeasure absorbance at 420 nm, 0.1 ml of each fraction was diluted with 2ml of 0.5 mM phosphate buffer at pH 7.5. To measure electricconductivity, each fraction was diluted with distilled water to 0.5%. Asucrose content was measured by HPLC.

To examine relationship of an anti-virus activity to each of thechromatographic peaks, the fractions corresponding to the peak of theabsorbance at 420 nm were grouped into 4 samples; those corresponding tothe sucrose peak into 3 samples; and those corresponding to the effluentlater than the sucrose peak into 1 sample. That is, fractions 3 and 4were combined together as sample 1; fractions 5 and 6 as sample 2;fractions 7 and 8 as sample 3; fractions 9 and 10 as sample 4; fractions11 and 12 as sample 5; fractions 13 and 14 as sample 6; fractions 15 and16 as sample 7; and fractions from 17 to 30 as sample 8. Fractions 1and2 were discarded since almost nothing was eluted. Each sample wasfreeze-dried overnight to become powder. Zero point twenty five gram ofthe freeze-dried powder obtained was dissolved in 0.5 mM phosphatebuffer at pH7.5 to a total volume of 100 ml of which absorbance at 420nm was determined. The results are as shown in Table 1. Sample 8 had arelatively high absorbance of 0.86, because it was a collection of thetail of the peak. While the samples other than sample 8 were each amixture of two fractions, sample 8 was a mixture of 14 fractions.Therefore, sample 8 has a higher absorbance, but it is not efficient forthe present purpose to collect those fractions only. In Table 1, theelectric conductivity ash content was calculated from a factordetermined from a correlation of electric conductivities with knownsulfate ash contents.

The analytical results are as shown in Table 1. In the table, adistribution ratio of a freeze-dried solid content means a weight ratioof a solid content of each sample to a total solid content of the entiresamples. The electric conductivity ash content and the content of eachsaccharide are ratios of those to a solid content of each sample.

Judging from each of the saccharides contents, it is seen that samples 1to 3 correspond to a fraction with less sugar and samples 4 to 8 to asugar-containing fraction. TABLE 1 Analytical Data of 2nd MolassesSamples Fractionated with an Ion Exchange Resin Electric Distributionratio of a conductivity Sucrose Glucose Fructose freeze-dried solid ashcontent content content content content (%) (%) (%) (%) (%) AbsorbanceSample 1 3.1 32.9 0 0 0 2.72 Sample 2 7.2 39.7 0 0 0 2.01 Sample 3 10.751.4 10.4 0 0 0.83 Sample 4 21.9 32.6 45.6 1.4 0.7 0.33 Sample 5 25.918.8 64.2 2.9 1.3 0.16 Sample 6 19.6 10 73.5 4.7 2.3 0.11 Sample 7 8.43.5 74 6.2 3.2 0.14 Sample 8 3.1 3.3 29.8 4.5 4.4 0.86 Separated liquidby — 43.7 5.9 0.9 1.4 1.04 Ion chromatography

Preparation Example 7 Desalinization of the Extract Obtained by IonChromatographic Separation

Using an electrodialysis bath, CH-0, ex Asahi Glass Co., provided with acation exchange membrane, Selemion CMV, and an anion exchange membrane,Selemion AMV, both ex Asahi Glass Co., the sugar cane-derivedextract(condensed liquid extract) prepared in Preparation Example 5 wasdesalinized by electrodialysis.

Ten liters of a 100 g/l sodium chloride solution as a condensingsolution, and 4.0 liters of a 50 g/l sodium sulfate solution as anelectrode solution were used. As a raw material, 10.7 liters of the ionchromatography effluent was used.

Operating conditions were as follows. An electric voltage was keptconstant at 3.0V. Initially, an electric current was 8.15 A, whichgradually decreased as desalinization proceeded. When 5 hours passed,the electric current was 1.6 A, 8 liters of the condensing liquid weretaken out and 8 liters of water was added for dilution. Subsequently,the operation was resumed and continued for 7 hours in total. The finalelectric current was 0.6 A. Progress of the desalinization was monitoredby measuring chloride ion and sulfate ion concentrations in thedesalinized liquid with time. In the desalinization by electrodialysis,equivalent amounts of anion and cation are removed. Cations such aspotassium ion, sodium ion, calcium ion, and magnesium ion are removedalmost equally regardless of ion species, but anions such as chlorideion and sulfate ion are removed on a different ratio. In this Example,concentrations of chloride ion and sulfate ion per dried solid, astypical anions, were determined by HPLC and their removal ratios werecalculated. At the beginning of the desalinization, a content ofchloride ion per dried solid of the extract to be desalinized was 5.45wt %, that of sulfate ion was 7.41wt %, and that of sulfate salts was43.0 wt %. After completion of the desalinization, a content of chlorideion per dried solid was 0.03 wt % (removal ratio of 99.4%), that ofsulfate ion was 6.61 wt % (removal ratio of 10.8%), and that of sulfatesalts was 34.7 wt % (removal ratio of 19.3%).

By electrodialysis, only a little quantity of sulfate ion was removed,but most of chloride ion was removed. The obtained extract wasfreeze-dried overnight to become powder. Zero point twenty five gram ofthe obtained powder was dissolved in 0.5 mM phosphate buffer at pH7.5 toa total volume of 100 ml of which absorbance at 420 nm was measured. Theabsorbance was 1.26.

Test Example 1 Acute Toxicity Test of Sugar Cane-Derived Extract

Using the extract powder prepared in Preparation Example 1, a toxicitytest by single oral administration was carried out using rats. Sixteenmale and sixteen female Sprauge-Dawley line SPF rats (Crj:CD(SD)) of 5week-old were quarantined and fed for about a week. Healthy rats wereselected and subjected to the test at 6 week-age. At the time ofadministration, the male rats weighed from 157 to 171 g and the femalerats from 123 to 133 g. The rats were fed in an animal room lit for 12hours at a temperature of 23+/−3° C., a relative humidity of 50+/−2%,and a ventilation frequency of 10 to 15 times per hour, in which therats were allowed to freely intake solid food, CRF-1, trade name, exOriental Yeast Co., and drinking water. To the rats fasted overnight(for about 16 hours) before the day of the administration, the sugarcane-derived extract powder of a predetermined concentration wasforcibly administered orally one time at 10 ml/kg body weight. To acontrol group, sterilized distilled water only was administered in thesimilar manner. Feeding was resumed 6 hours after the administration.

In addition to the control group, there were 2 groups with differentdosages of 200 mg/kg and 1000 mg/kg. Thus, 3 groups in total weretested. Each group consisted of 5 males and 5 females. The results areas shown in Table 2. TABLE 2 Toxicity Test by a Single OralAdministration Concentration Administered volume the number the number(weight/volume %) (ml/kg body weight) sex of rats of deaths animal No. 010 male 5 0/5 1001˜1005 0 10 female 5 0/5 1101˜1105 2 10 male 5 0/52001˜2005 2 10 female 5 0/5 2101˜2105 10 10 male 5 0/5 3001˜3005 10 10female 5 0/5 3101˜3105

A lethal dose is estimated to be larger than 1000 mg/kg, because neitherfemale nor male rat died 14 days after the administration of the maximumdosage of 1000 mg/kg.

During the feeding of the rats, no abnormality was observed in any ofthe rats. In addition, the dosed groups showed almost identical changein body weight with that of the control group, and their increase inbody weight during the observation period is almost the same as that ofthe control group. In an anatomical examination, no rat showedabnormality in organs and tissues in the body surface, head, chest, andabdominal region.

Judging from the aforesaid results, toxicity of the extract powderobtained in Preparation Example 1 is extremely low, when the powder isorally administered to a rat one time.

Test Example 2 Antibacterial Activity of the Sugar Cane-Derived ExtractAgainst E. coli

Using each of the extract powders prepared in Preparation Examples 1 to4, minimum growth inhibiting concentrations (MIC, μg/ml) for 6 strainsof Escherichia coli (E. coli) were determined according to the methodspecified by the Japanese Chemotherapy Association. The extract wasdissolved in and diluted with sterilized distilled water to five levelsof concentration of 100 μg/ml, 300 μg/ml, 1000 μg/ml, 3000 μg/ml, and10000 μg/ml and MIC was determined. MIC was found to be 1000 μg/ml forall of the 6 strains of E. coli, so that no strong antibacterialactivity was observed. The results are as shown in Table 3 below. TABLE3 Minimum Growth Inhibiting Concentration for Each of the Bacteria(μg/ml) Exatract in Exatract in Exatract in Prep. Prep. Prep. Exatractin Name of bacteria Example 1 Example 2 Example 3 Prep. Example 4Escherichia coli 10000 10000 10000 10000 NIHJ Escherichia coli 1000010000 10000 10000 C-1 Escherichia coli 10000 10000 10000 10000 C-2Escherichia coli 10000 10000 10000 10000 C-3 Escherichia coli 1000010000 10000 10000 TK-18A Escherichia coli 10000 10000 10000 10000 E01292

Similar tests were carried out for several strains of other bacteria,yeast and fungus. MICs for bacteria (Pseudomonas aureofaciens), yeasts(Saccharomyces cerevisiae, Hansenula anomala, etc.) and fungi(Chaetomium globsum) were 1000 μg/ml, which indicates a strongerantibacterial activity than that against E. coli.

Test Example 3 Proliferation-Inhibiting Property of the SugarCane-Derived Extract

Using each of the extract powders prepared in Preparation Examples 1 and2, a proliferation inhibiting-property against Coxsackie virus type B6Schmitt strain and Herpes simplex virus type 1 HF strain was examined.

At first, cytotoxicity of the extract to human embryo lung-derived cell(HEL-R66 cell) was examined. The sugar cane-derived extract wasdissolved in and diluted with sterilized distilled water to aconcentration of from 125 to 2000 μg/ml and applied to cultured cells.After 4 day-long culture, existence of denaturation in the cells wasobserved with a microscope. As shown in Table 4, no toxicity to thecells was observed up to a concentration of 1000 μg/ml.

Next, 100 PFU of viruses were inoculated to the cells. After the viruseswere adsorbed to the cells, the excess viruses were removed. To amaintenance medium for the cells, the sugar cane-derived extract wasadded in such an amount that a final concentration of the extract was inthe range of from 125 to 1000 μg/ml. The cells with the viruses adsorbedthereto were cultured for 4 days. After the cultivation, existence ofproliferation of the cells was observed with a microscope. As shown inTables 5 and 6, it was found that the sugar cane-derived extract doesnot have a proliferation-inhibiting effect against Coxsackie virus,while it has a proliferation-inhibiting effect against Herpes virus at aconcentration of from 500 to 1000 μg/ml. TABLE 4 Cytotoxicity Finalconcentration of sugar cane-derived extract (μg/ml) 125 250 500 10002000 Extract in − − − − + Preparation Example 1 Extract in − − − − +Preparation Example 2 Sterilized distilled water − − − − − (control)

TABLE 5 Proliferation Inhibiting-Property against Coxsackie Virus TypeB6 Schmitt Strain Final concentration of sugar cane-derived extract(μg/ml) 125 250 1000 Extract in − − − Preparation Example 1 Extract in −− − Preparation Example 2 Sterilized distilled water − − − (control)

TABLE 6 Proliferation Inhibiting-Property against Herpes Simplex VirusType 1HF Strain Final concentration of sugar cane-derived extract(μg/ml) 125 250 500 1000 Extract in − − ± + Preparation Example 1Extract in − − ± + Preparation Example 2 Sterilized distilled water − −− − (control)

Example 1

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group. Each of the extract prepared in the aforesaidPreparation Examples 1 to 4 was dissolved or suspended in sterilizeddistilled water. The extract solution or suspension was orallyadministered to the mice in an amount of 100 mg/kg or 500 mg/kg on theday before inoculation of E. coli. To a control group of the mice, thesame volume of sterilized distilled water was orally administered. Asuspension of man-origin E. coli was subcutaneously inoculated to themice in an amount of 0.2 ml which corresponded to 1 minimum lethal dose,MLD, (4.0×10⁷ CFU/mouse). Four days' survival ratio was determined 4days after the inoculation. The results were evaluated by χ² test asshown in Table 7. TABLE 7 Infection Preventing Test against E. coliAdministered amount Survival ratio Tested Exatract of extract (%) χ²test Control 0 Exatract in Preparation 100 mg/kg 40 * Example 1 Exatractin Preparation 200 mg/kg 60 ** Example 1 Exatract in Preparation 500mg/kg 80 ** Example 1 Exatract in Preparation 100 mg/kg 30 Example 2Exatract in Preparation 500 mg/kg 50 * Example 2 Exatract in Preparation100 mg/kg 30 Example 3 Exatract in Preparation 200 mg/kg 50 * Example 3Exatract in Preparation 500 mg/kg 80 ** Example 3 Exatract in Preventive100 mg/kg 0 Example 4 Exatract in Preventive 200 mg/kg 10 Example 4Exatract in Preventive 500 mg/kg 40 * Example 4* p < 0.05,** p < 0.01

The groups which were inoculated with E. coli after the administrationof the sugar cane-derived extract clearly showed higher survival ratios,which ratio increased with the increasing amount of the administration.That is, an infection preventing effect was recognized in the sugarcane-derived extract.

Example 2

1) Tested Extracts

In addition to the sugar cane-derived extracts prepared in PreparationExamples 1 to 4, the following extract was also prepared. Thus, firstextract prepared in Preparation Example 2 was suspended in sterilizeddistilled water and dialyzed against sterilized water in a dialysis tubemade of cellulose ester with a fractionating molecular weight of 1000(Spectra/Por, tradename, cellulose ester membrane MWCO:1000, ex SpectrumCo.). The resultant liquid inside the dialysis membrane which isreferred to as the fraction with a molecular weight of 1000 or more, andthe liquid outside the membrane which is referred to as the fractionwith a molecular weight smaller than 1000, were condensed to dryness andused for tests.

2) Anti-Virus Test

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group. Each of the extract prepared in Preparation Examples1 to 4, the fraction with a molecular weight of 1000 or more, and thefraction with a molecular weight smaller than 1000 from PreparationExample 2 was dissolved or suspended in sterilized distilled water. Theextract solution or suspension was orally or intramuscularlyadministered to the mice in an amount shown in Table 7. Theadministration was performed 3 times in total, i.e., immediately after,one day after and two days after the inoculation of virus, or 9 times intotal, i.e., three times per day×3 consecutive days. To the referencegroup of the mice, the same volume of sterilized distilled water wasorally administered. A suspension of pig-origin Pseudorabies virus wassubcutaneously inoculated to the mice in an amount of 0.2 ml, whichcorresponded to 1 MLD (133 PFU/mouse). Seven days' survival ratio wasdetermined. The results were evaluated by χ² test as shown in Table 8.TABLE 8 Infection Preventing Test against Virus AdministeredAdministration The number of Survival Tested Extract amount of extractmethod administration ratio (%) χ² test Control Oral 0 Exatract in Prep.Ex. 1 100 mg/kg Oral ×3*¹ 20 Exatract in Prep. Ex. 1 200 mg/kg Oral ×360 ** Exatract in Prep. Ex. 1 500 mg/kg Oral ×3 80 ** Exatract in Prep.Ex. 2 100 mg/kg Oral ×3 10 Exatract in Prep. Ex. 2 200 mg/kg Oral ×3 30Exatract in Prep. Ex. 2 500 mg/kg Oral ×3 70 ** Exatract in Prep. Ex. 3100 mg/kg Oral ×3 20 Exatract in Prep. Ex. 3 200 mg/kg Oral ×3 40 *Exatract in Prep. Ex. 3 500 mg/kg Oral ×3 70 ** Exatract in Prep. Ex. 4100 mg/kg Oral ×3 10 Exatract in Prep. Ex. 4 200 mg/kg Oral ×3 30Exatract in Prep. Ex. 4 500 mg/kg Oral ×3 60 ** Exatract in Prep. Ex. 2 25 mg/kg Oral ×3 × 3*² 0 Exatract in Prep. Ex. 2  50 mg/kg Oral ×3 × 330 Exatract in Prep. Ex. 2 100 mg/kg Oral ×3 × 3 40 * Exatract in Prep.Ex. 2 200 mg/kg Oral ×3 × 3 70 ** Exatract in Prep. Ex. 2 500 mg/kg Oral×3 × 3 90 ** Exatract in Prep. Ex. 2 125 mg/kg Oral ×3 20 (Mw <1000)Exatract in Prep. Ex. 2 250 mg/kg Oral ×3 40 * (Mw <1000) Exatract inPrep. Ex. 2 500 mg/kg Oral ×3 80 ** (Mw <1000) Exatract in Prep. Ex. 2500 mg/kg Oral ×3 20 (Mw >= 1000) Exatract in Preparation 1.56 mg/kg Intramuscular ×3 20 Example 2 (Mw <1000) Exatract in Prep. Ex. 2 6.25mg/kg  Intramuscular ×3 30 (Mw <1000) Exatract in Prep. Ex. 2 25.0mg/kg  Intramuscular ×3 60 ** (Mw <1000)* p < 0.05,** p < 0.01*¹Administered one time per day × 3 consecutive days.*²Administered three times per day × 3 consecutive days.

The groups administered the sugar cane-derived extract clearly showedhigher survival ratios which increased with the increasing amount of theadministration. That is, a preventive effect for infection wasrecognized in the sugar cane-derived extract. In the processed extractfrom Preparation Example 2, the fraction with a molecular weight smallerthan 1000 had a higher effect than the fraction with a molecular weightof 1000 or higher. A significant survival ratio was attained by theintramuscular administration. Thus, it is found that the present sugarcane-derived extract is effective also when intramuscularly administeredbased on the fact that.

Example 3 Infection-Preventing Effect Against E. coli by an Intake ofthe Less-Succharaide Fraction

1) Tested Extract

The extract powder prepared by ion chromatographic separation inPreparation Example 5 and the desalinized powder of extract prepared byion chromatographic separation in Preparation Example 7 were tested.

2) Test of a Preventive Effect Against Infection by E. coli

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group.

Each of the aforesaid extract was dissolved or suspended in sterilizeddistilled water and orally administered in an amount shown in Table 9 tothe mice on the day before the inoculation of E. coli. To the controlgroup of the mice, the same volume of sterilized distilled water wasorally administered. A suspension of man-origin E. coli wassubcutaneously inoculated to the mice in an amount of 0.2 ml whichcorresponded to 1 MLD (4.0×10⁷ CFU/mouse). Four days' survival ratio wasdetermined. The results evaluated by χ² test are as shown in Table 9.

The groups administered the sugar cane-derived extract clearly showedhigher survival rates which increased with increasing amount of theadministration. No effect of desalinization was observed. TABLE 9Infection Preventing Test against E. coli Administered amount ofnon-sugar content extract Survival ratio Tested Exatract (%) mg/kg (asnon-sugar) (%) χ² test Control Sterilized water 0.5 ml 0 Exatract inPreparation 91.8 109 (100*¹) 20 Example 5 Exatract in Preparation 91.8218 (200) 50 * Example 5 Exatract in Preparation 91.8 545 (500) 70 **Example 5 Exatract in Preparation 88.0 114 (100) 10 Example 7 Exatractin Preparation 88.0 227 (200) 40 * Example 7 Exatract in Preparation88.0 568 (500) 70 ** Example 7*¹In Tables here in after, numerals in parentheses are contents ofnon-sugar only.

Example 4 Anti-Virus Effect by an Intake of the Less-Sugar Fraction

1) Tested Extracts

Total 10 extracts were tested: the powder of the extract prepared inPreparation Example 5, the powders of samples 1 to 8 prepared inPreparation Example 6 and the powder of desalinized extract prepared byion chromatographic separation in Preparation Example 7.

2) Anti-Virus Test

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group.

Each of the aforesaid extract was dissolved or suspended in sterilizeddistilled water and orally, forcedly administered in an amount shown inTable 10 to the mice. The administration was performed 3 times in total,i.e., immediately after, one day after and two days after theinoculation of virus. To the control group of the mice, the same volumeof sterilized distilled water was orally forcedly administered. Asuspension of pig-origin Pseudorabies virus was subcutaneouslyinoculated to the mice in an amount of 0.2 ml which corresponded to 1MLD (133 PFU/mouse). Seven days' survival ratio was determined. Theresults evaluated by χ² test are as shown in Table 10.

The groups administered the sugar-cane-derived extract clearly showedhigher survival ratios which increased with the increasing amount of theadministration.

The extracts having a higher non-sugar content, i.e., the extract ofPreparation Example 5, the extracts of samples 1 to 3 of PreparationExamples 6, and the extract of Preparation Example 7, showedparticularly higher survival ratios. Therefore, sugar is not consideredto be the present active ingredient. TABLE 10 Infection Preventing Testagainst Virus Administered amount of non-sugar extract mg/kg (as non-Survival ratio Tested Extract content (%) sugar) (%) χ² test ControlSterilized water 0.5 ml 0 Exatract in Preparation 91.8 109 (100) 10Example 5 Exatract in Preparation 91.8 218 (200) 40 * Example 5 Exatractin Preparation 91.8 545 (500) 80 ** Example 5 Exatract sample 1 in 100.0200 (200) 40 * Preparation Example 6 Exatract sample 1 in 100.0 500(500) 80 ** Preparation Example 6 Exatract sample 2 in 100.0 200 (200)50 * Preparation Example 6 Exatract sample 2 in 100.0 500 (500) 90 **Preparation Example 6 Exatract sample 3 in 89.6 223 (200) 30 PreparationExample 6 Exatract sample 3 in 89.6 558 (500) 80 ** Preparation Example6 Exatract sample 4 in 52.3 382 (200) 20 Preparation Example 6 Exatractsample 4 in 52.3 956 (500) 60 ** Preparation Example 6 Exatract sample 5in 31.6 1582 (500)  50 * Preparation Example 6 Exatract sample 6 in 19.52564 (500)  40 * Preparation Example 6 Exatract sample 7 in 16.6 3012(500)  40 * Preparation Example 6 Exatract sample 8 in 61.2 327 (200) 30Preparation Example 6 Exatract sample 8 in 61.2 817 (500) 60 **Preparation Example 6 Exatract in Preparation 89.2 112 (100) 20 Example7 Exatract in Preparation 89.2 224 (200) 40 * Example 7 Exatract inPreparation 89.2 561 (500) 70 ** Example 7

Test Example 4 Fractions Separated with Sephadex G-25, Based onMolecular Weight, from the Extract Liquid Separated by IonChromatography and Bagasse Extract and Anti-Virus Effect Thereof

Gel permeation chromatography was performed on the extract prepared inPreparation Example 3 (hot water extraction of bagasse) and the extractprepared in Preparation example 5 (liquid extract prepared by ionchromatography) for fractionation based on molecular weight.

The aforesaid extracts were pre-treated to prevent precipitates possiblypresent in the extract from clogging. The extract of Preparation Example3 was diluted to a Brix of from 17.5 to 17.8 with distilled water andcentrifuged at 600×g for 15 minutes to remove insoluble materials. Thesupernatant was suction filtered through a No. 2 filter paper, exAdvantec Toyo Co., or a glass fiber filter paper GA55, ex Advantec ToyoCo. The filtrate was subjected to gel permeation chromatography. Theextract of Preparation Example 5 was diluted to a Brix of from 18.7 to22.2 with distilled water and filtered through a glass fiber filterpaper GA55, ex Advantec Toyo Co. The filtrate was subjected to gelpermeation chromatography.

A column having an inner diameter of 26 mm and a height of 630 mm waspacked with 315 ml of Sephadex G-25 Superfine, trade name of AmashamPharmacia Biotech Co. FPLC system, ex Pharmacia Co was used forchromatography.

A degassed solution of ethanol/water=35/65 by volume was used as aneluent, which was passed through the column at a flow rate of SV=0.25hr⁻¹ (1.32 ml/min) at a room temperature. For the extract of PreparationExample 3, a sample feeding amount was 6 ml when filtered with No. 2filter paper, and 17 ml when filtered with the glass fiber filter paper.For the extract of Preparation Example 5, 6 ml was fed. Thechromatography was repeated at least 5 times in the same conditions toconfirm reproducibility in chromatogram. At about 80 minutes after thesample feeding was initiated, collection was started. One fractionationwas collected over 15 minutes. A total of 20 fractions were collectedfrom each of the extracts of Preparation Examples of 3 and 5.Chromatograms are as shown in FIGS. 4 and 5. FIG. 6 shows a chromatogramof a molecular weight marker in the same conditions.

The twenty fractions were combined into 3 samples: sample 1 consistingof fractions 1 to 4 containing substances with molecular weight of 10000or more, sample 2 consisting of fractions 5 to 11 up to a front part ofthe peak in electric conductivity, and sample 3 consisting of fractions12 to 20 containing much salts.

These samples 1 to 3 were freeze-dried to become powder. Analysisresults are as shown in Table 11. Here, the definitions of adistribution ratio of freeze-dried solid content and saccharidescontents are the same as those in Table 1. Using each of the powders, ananti-virus test was made in the same manner as in Example 4. The resultsare as shown in Table 12.

No significant differences among samples 1 to 3 were observed. Fromthis, it is seen that there are a plurality of anti-virus activesubstances both in the extracts obtained by ion chromatographicseparation and in the hot water extract of bagasse, which substanceshave a wide range of molecular weights. TABLE 11 Distribution ratio offreeze-dried Sucurose Glucose Fructose solid content content contentcontent (%) (%) (%) (%) Fractionated sample 1 in 8.8 0 0 0 PreparationExample 3 Fractionated sample 2 in 42.3 20.7 3.5 3.1 Preparation Example3 Fractionated sample 3 in 48.9 0 0 0 Preparation Example 3 Fractionatedsample 1 in 46.5 0 0 0 Preparation Example 5 Fractionated sample 2 in28.8 9.8 1.9 1.6 Preparation Example 5 Fractionated sample 3 in 24.8 0 00 Preparation Example 5

TABLE 12 Infection Preventing Test against Virus Administered amount ofNon-sugar extract mg/kg (as non- Survival ratio Tested extract content(%) sugar) (%) χ²test Control Sterilized water 0.5 ml 0 Fractionatedsample 1 in 100.0 200 (200) 40 * Preparation Example 3 Fractionatedsample 1 in 100.0 500 (500) 90 ** Preparation Example 3 Fractionatedsample 2 in 72.7 28 (20) 30 Preparation Example 3 Fractionated sample 2in 72.7 688 (500) 70 ** Preparation Example 3 Fractionated sample 3 in100.0 200 (200) 30 Preparation Example 3 Fractionated sample 3 in 100.0500 (500) 80 ** Preparation Example 3 Fractionated sample 1 in 100.0 200(200) 50 * Preparation Example 5 Fractionated sample 1 in 100.0 500(500) 90 ** Preparation Example 5 Fractionated sample 2 in 86.7 231(200) 30 Preparation Example 5 Fractionated sample 2 in 86.7 577 (500)70 ** Preparation Example 5 Fractionated sample 3 in 100.0 200 (200)50 * Preparation Example 5 Fractionated sample 3 in 100.0 500 (500) 90** Preparation Example 5

Example 5 Evaluation of Effects of Vaccine Adjuvants

1) Tested Extracts

The following extracts were tested: the extract powder prepared bycolumn chromatography in Preparation Example 1, the extract powderprepared from bagasse in Preparation Example 3, the extract powderprepared by ion chromatography in Preparation Example 5 and the extractpowder prepared by desalinizing the extract obtained by ionchromatography in Preparation Example 7.

2) Evaluation of Effects of Vaccine Adjuvants

S1c:ICR mice of 5 week-old (male, about 30 g in body weight) were usedin 10 mice per group.

Effects of vaccine adjuvants were tested in administration of thevarious sugar cane-derived extracts to the mice.

For the groups administered the extracts of Preparation Examples 1, 3,5, and 7, commercially available pig-origin Pseudorabies virus vaccine(AWV) was diluted with a physiological saline solution about 20 timesand 0.2 ml of the suspension was intramuscularly administered to themice. Each extract powder in an amount of 500 mg less-sugar content/kgwas dissolved in 0.5 ml of sterilized water. The extract solution wasorally administered to the mice once per day for 6 days starting fromthe day of the vaccination. Fourteen days after the vaccination, 0.2 mlof a suspension of pig-origin Pseudorabies virus diluted with aphysiological saline solution corresponding to 1 MLD was subcutaneouslyinoculated to the mice and 7 days' survival ratio was determined.

As to the extract of Preparation Example 3, additional group of mice wasused where a mixture of the vaccine and a solution prepared bydissolving the extraction in an amount of 100mg less non-sugarcontent/kg in 0.5 ml of sterilized water was administered.

To the group without vaccination, 0.2 ml of a physiological salinesolution instead of the vaccine and 0.5 ml of sterilized water insteadof the extract were administered. To the group without administration ofthe extract, 0.5 ml of sterilized water was administered instead of theextract.

Effects of the adjuvant were evaluated by χ² test against the survivalratio of the group without administration of the extract (with vaccineonly was administered). The results are as shown in Table 13.

No significant difference was observed between the group without vaccineadministration and the group without extract administration. The groupwhere the mixture of the extract and the vaccine was intramuscularlyadministered showed no significant difference in a survival ratio. Tothe contrary, in the groups where the sugar cane-derived extracts wereorally administered, significant increases in survival ratios wereobserved, which indicates that the present extract is effective as anvaccine adjuvant. TABLE 13 Evaluation of Effects of Vaccine Adjuvantsnon-sugar Administered amount of content extract mg/kg (as non-Administration Survival χ² Tested extract (%) sugar) method of extractImmunity ratio (%) test No inoculation Sterilized water 0.5 ml Oraladmistration Physiological 0 of vaccine saline solution 0.2 m 1 im. NoSterilized water 0.5 ml Oral admistration AWV 0.2 m 1 im. 20administration of extract Exatract of 96.0 521 (500) Oral admistrationAWV 0.2 m 1 im. 80 * Prep. Ex. 1 Exatract of 87.7 570 (500) Oraladmistration AWV 0.2 m 1 im. 80 * Prep. Ex. 3 Exatract of 91.8 545 (500)Oral admistration AWV 0.2 m 1 im. 80 * Prep. Ex. 5 Exatract of 89.2 561(500) Oral admistraion AWV 0.2 m 1 im. 70 * Prep. Ex. 7 Exatract of 87.7114 (100) Intramuscular AWV 0.2 m 1 im. 20 Prep. Ex. 3 administration*¹*¹Mixture with vaccine was admistered.AWV: Pseudorabies virus vaccine.im.: Intramuscular administeration*: 0.01 < p ≦ 0.05

Example 6

1) Tested Extracts

The following extracts were tested; the extract powder prepared inPreparation Examples 1 to 4 and the extract powders of PreparationExample 2 processed in the same manner as in Example 2, i.e., thefraction with a molecular weight of 1000 or smaller, and the fractionwith a molecular weight larger than 1000.

2) Anti-Endotoxin Effect

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group.

The extracts of Preparation Examples 1 to 4 and the processed extract ofPreparation Example 2 were each dissolved or suspended in sterilizeddistilled water and orally administered to the mice in an amount of 100mg/kg twice, one day before and 6 hours after injection of endotoxin,lipopolysaccharide, referred to as LPS hereinafter. To the control groupof mice, the same volume of sterilized distilled water was orallyadministered. E. coli-origin LPS was injected to a caudal vein of themice in an amount of 0.2 ml corresponding to the minimum lethal dosagefor attack by endotoxin. Four days' survival ratio was determined andevaluated by χ² test. The results are as shown in Table 14. TABLE 14Evaluation of Anti-endotoxin Shock Effect Survival Tested extract ratio(%) χ² test Control 0 Exatract in Preparation Example 1 50 * Exatract inPreparation Example 2 40 * Exatract in Preparation Example 3 40 *Exatract in Preparation Example 2 (Mw < 1000) 60 ** Exatract inPreparation Example 2 (Mw >= 1000) 20* p < 0.05,** p < 0.01

The group administered the sugar cane-derived extract showedsignificantly higher survival ratios, which indicates that the presentextract has an anti-endotoxin effect. For the processed extracts fromPreparation Example 2, the lower molecular weight fraction had an highereffect than the higher molecular weight fraction.

Example 7 Anti-Endotoxin Effect

1) Tested Extracts

The powder extract prepared by ion chromatographic separation inPreparation Example 5 and the powder extract desalinized by ionchromatographic separation in Preparation Example 7 were tested.

2) Anti-Endotoxin Effect

S1c:ICR male mice of 5 week-old (about 30 g in body weight) were used in10 mice per group.

Each of the extracts was dissolved or suspended in sterilized distilledwater and orally administered to the mice in an amount of 100 mg/kgtwice, on the day before and 6 hours after injection of endotoxin (LPS).To the control group of mice, the same volume of sterilized distilledwater was orally administered. E. coli-origin LPS was injected to acaudal vein of the mice in an amount of 0.2 ml corresponding to theminimum lethal dosage for attack by endotoxin. Four days' survival ratiowas determined and evaluated by χ² test. The results are as shown inTable 15. TABLE 15 Evaluation of Anti-endotoxin Shock Effect Amount ofNon-sugar administered Survival content extract ratio Tested extract (%)(mg/kg) (%) χ² test Control 0 Exatract in Prep. Example 5 91.8 100 50 *Exatract in Prep. Example 7 89.2 100 40 *

Example 8

Slc:ICR male mice of 3 week-old (about 12 g in body weight) were used in5 mice per group. Group 1 for control was fed with MF standard feed andgroups 2 to 5 were fed with MF standard feed added 0.1% of one of theextracts of Preparation Examples 1 to 4. At the end of 28 days feeding,a body weight was measured. Also, blood was collected and plasma wassubjected to biochemical analyses. The results of weight increase are asshown in Table 16 and those of the biochemical analyses are as shown inTable 17.

In Table 16, “weight increase” means weight gain in the test period;“weight increase ratio” means a ratio of the weight increase to theweight (g) at the beginning of the test; and “ratio of the weightincrease ratio” is percent of the weight increase ratio to the weightincrease ratio that of the control group. TABLE 16 Growth PromotingEffect of Exatract Weight Weight increase Ratio of weight Test groupincrease (g) ratio increase ratio (%) Control 27.6 2.29 100 Exatract inPrep. 31.4 2.60 114 Example 1 Exatract in Prep. 30.9 2.55 111 Example 2Exatract in Prep. 30.0 2.51 110 Example 3 Exatract in Prep. 29.9 2.48108 Example 4

The groups where the sugar cane-derived extract was fed showedsignificant increase in weight, which indicates a growth promotioneffect of the present extract. No abnormality was detected in thebiochemical analyses. TABLE 17 Biochemical Analyses Results of Plasmaafter Administration of Extract Analysis item GPT GOT ALP GLU CRNN T.CHOTest group (IU/L) (IU/L) (IU/L) (mg/dl) (mg/dl) (mg/dl) Control 35.4 ±49.8 50.8 ± 42.1 205.8 ± 23.3 156.0 ± 13.2 0.162 ± 0.028 114.6 ± 13.8Extract in 22.0 ± 10.5 46.0 ± 6.8 190.0 ± 39.0 179.0 ± 25.0 0.112 ±0.031 114.8 ± 15.3 Prep. Exmple 1 Extract in 31.0 ± 21.8 48.0 ± 9.7186.6 ± 28.8 181.2 ± 21.9 0.134 ± 0.029 115.4 ± 13.4 Prep. Exmple 2Extract in 16.4 ± 4.4 37.0 ± 8.8 172.2 ± 20.8 170.8 ± 10.0 0.116 ± 0.010103.8 ± 15.8 Prep. Exmple 3 Extract in 18.6 ± 4.5 53.8 ± 23.3 220.4 ±35.5 190.0 ± 24.4 0.120 ± 0.032 110.0 ± 12.6 Prep. Exmple 4 Analysisitem TG T.PRO PL ALB-U LDH Test group (mg/dl) (g/dl) (mg/dl) (g/dl)(IU/L) Control 91.6 ± 46.1 4.22 ± 0.34 225.2 ± 27.3 2.44 ± 0.20 962 ±993 Extract in 112.8 ± 35.0  4.42 ± 0.17 220.6 ± 20.7 2.56 ± 0.10 968 ±259 Prep. Exmple 1 Extract in 90.4 ± 25.4 4.40 ± 0.27 218.2 ± 17.9 2.54± 0.22 1113 ± 277  Prep. Exmple 2 Extract in 88.8 ± 19.4 4.04 ± 0.08202.2 ± 15.9 2.40 ± 0.00 767 ± 204 Prep. Exmple 3 Extract in 81.2 ± 33.53.88 ± 0.16 202.1 ± 17.9 2.26 ± 0.10 1420 ± 747  Prep. Exmple 4

Example 9 Growth Promoting Effect of the Extracts from PreparationExamples 3, 5 and 7

Slc:ICR male mice of 3 week-old (about 12 g in body weight) were used in5 mice per group. A dosage of non-sugar portion was made constant,because sugar was not considered to be active ingredient. A controlgroup was fed with MF standard feed and test groups were freely given MFstandard feed mixed with 0.1% (as non-sugar content) of one of theextracts from Preparation Example 3 (bagasse extract), Example 5(extractseparated by ion chromatography) and Example 7 (extract separated by ionchromatography and desalinized. At the end of 28 days feeding, a bodyweight was measured. Also, blood was collected and plasma was subjectedto biochemical analyses. The results of weight increase are as shown inTable 18 and those of the biochemical analyses are as shown in Table 19.

In Table 18, “weight increase” means weight gain in the test period;“weight increase ratio” means a ratio of the weight increase to theweight (g) at the beginning of the test; and “ratio of the weightincrease ratio” is percent of the weight increase ratio to the weightincrease ratio that of the control group. TABLE 18 Growth PromotingEffect of Extract Weight Weight increase Ratio of weight Test groupincrease (g) ratio increase ratio (%) Control 27.1 2.34 100 1.00%Addition of extract in 29.3 2.54 109 Preparation Example 3 (0.1%addition as non-sugar components) 0.109% Addition of extract in 29.52.52 108 Preparation Example 5 (0.1% addition as non-sugar components)0.114% Addition of extract 28.9 2.49 106 in Preparation Example 7 (0.1%addition as non-sugar components)

TABLE 19 Biological Analyses Results of Plasma after Administration ofExatract Analysis item GPT GOT ALP GLU CRNN T.CHO Test group (IU/L)(IU/L) (IU/L) (mg/dl) (mg/dl) (mg/dl) Control 25.8 ± 5.7 53.6 ± 14.8175.6 ± 90.7 164.8 ± 33.5 0.114 ± 0.021 116.2 ± 11.2 Addition of Extract28.4 ± 5.2 55.2 ± 14.8 194.6 ± 23.8 215.8 ± 36.6 0.132 ± 0.024 102.4 ±12.1 in Prep. Exmple 3 Addition of Extract 91.6 ± 9.2 52.2 ± 13.8 186.4± 38.1 161.2 ± 29.8 0.132 ± 0.029  90.0 ± 15.9 in Prep. Exmple 5Addition of Extract 30.2 ± 6.3 61.6 ± 16.1 192.8 ± 14.6 210.0 ± 17.80.142 ± 0.040 98.8 ± 8.4 in Prep. Exmple 7 Analysis item TG T.PRO PLALB-U LDH Test group (mg/dl) (g/dl) (mg/dl) (g/dl) (IU/L) Control 113.8± 42.6  4.36 ± 0.24 218.0 ± 16.5 2.46 ± 0.10 973.0 ± 500   Addition ofExtract 36.2 ± 13.1 4.18 ± 0.07 197.0 ± 9.5  2.50 ± 0.11 456.0 ± 44.4 in Prep. Exmple 3 Addition of Extract 81.2 ± 42.4 4.18 ± 0.34 179.6 ±30.2 2.34 ± 0.22 956 ± 135 in Prep. Exmple 5 Addition of Extract 44.0 ±21.7 4.34 ± 0.10 208.2 ± 12.2 2.62 ± 0.07 849 ± 326 in Prep. Exmple 7

INDUSTRIAL APPLICABILITY

By administrating the present sugar cane-derived extract to man oranimals orally, infection by bacteria or viruses for example can beprevented or remedied. In addition, diseases caused by endotoxin can beprevented or remedied.

The present sugar cane-derived extract works as an vaccine adjuvant andalso promotes growth when administered orally, for example, to man oranimals. The sugar cane-derived extract is plant-origin and a naturalproduct which has been taken in by man from ancient times asnon-centrifugal sugar such as brown sugar (KOKUTOU) and, accordingly, issafe to human or animal health. Also, the extract can be produced in lowcosts. The present extract is a natural product, but has a highpreventive or remedial effect, an anti-endotoxin effect, a vaccineadjuvant effect, and a growth promoting effect even in a small dosage.

1-15. (canceled)
 16. A method for enhancing antigenicity of a vaccinewhen administered to a man or animal, comprising the step ofadministering to man or animal a vaccine adjuvant before, concurrentlywith, or after administration of the vaccine, wherein the vaccineadjuvant comprises a sugar cane-derived extract as an active ingredient.17. The method according to claim 16, wherein the sugar cane-derivedextract is a fraction obtained by treating a raw material selected fromthe group consisting of sugar cane juice, a liquid extract from sugarcane, and sugar cane-derived molasses, in column chromatography with afixed carrier.
 18. The method according to claim 17, wherein the sugarcane-derived extract is a fraction obtained by passing the raw materialselected from the group consisting of sugar cane juice, a liquid extractfrom sugar cane, and sugar cane-derived molasses, through a columnpacked with a synthetic adsorbent as the fixed carrier and elutingsubstances adsorbed onto the synthetic adsorbent with a solvent selectedfrom the group consisting of water, methanol, ethanol and mixturesthereof.
 19. The method according to claim 17, wherein the sugarcane-derived extract is a fraction which absorbs light of a wavelengthof 420 nm and is from fractions obtained by column chromatographictreatment utilizing differences in affinity to a fixed carrier which isan ion exchange resin packed in a column.
 20. The method according toclaim 19, wherein the ion exchange resin is a cation exchange resin. 21.The method according to claim 20, wherein the cation exchange resin is astrongly acidic cation exchange resin.
 22. The method according to claim21, wherein the strongly acidic cation exchange resin is of a sodium ionform or a potassium ion form.
 23. The method according to claim 19,wherein the ion exchange resin is a gel form resin.
 24. The methodaccording to claim 19, wherein ion exchange chromatographic separationis carried out using a pseudo moving-bed continuous separation method.25. The method according to claim 19, wherein the fraction absorbinglight of a wavelength of 420 nm is further treated by electrodialysis tothereby decrease amounts of salts contained in said fraction.
 26. Themethod according to claim 16, wherein the sugar cane-derived extract isobtained by a step of extracting bagasse with an extractant selectedfrom the group consisting of water, a hydrophilic solvent and mixturesthereof.
 27. The method according to claim 26, wherein the hydrophilicsolvent used during extraction is ethanol.
 28. The method according toclaim 26, wherein the solvent used during extraction is a mixture ofethanol and water in a volume ratio of 60/40 or lower.
 29. The methodaccording to claim 16, wherein the vaccine adjuvant is administered inthe form of a food, which comprises the sugar cane-derived extract. 30.The method according to claim 29, wherein the food is an animal feed.31-60. (canceled)
 61. The method according to claim 23, wherein ionexchange chromatographic separation is carried out using a pseudomoving-bed continuous separation method.
 62. The method according toclaim 23, wherein the fraction absorbing light of a wavelength of 420 nmis further treated by electrodialysis to thereby decrease amounts ofsalts contained in said fraction.
 63. The method according to claim 24,wherein the fraction absorbing light of a wavelength of 420 nm isfurther treated by electrodialysis to thereby decrease amounts of saltscontained in said fraction.